Proteomic analysis of retinal tissue in an S100B autoimmune glaucoma model

Glaucoma is not only the second leading cause of blindness worldwide, but patient numbers will also increase significantly in the coming years due to aging societies. Glaucoma is a progressive optic neuropathy with changes at the optic nerve head, gradual retinal ganglion cell (RGC) death, and visual field loss [1]. Unfortunately, glaucoma can remain asymptomatic until it is rather far progressed, hence about 10-50 % of patients are unaware they suffer from this disease. High intraocular pressure (IOP) is the main risk factor; however, normal-tension glaucoma (NTG) occurs in patients with physiological IOP [2]. This form account for about 30 % of glaucoma patients [3]. Furthermore, sustained IOP lowering can slow down, but does not completely stop disease progression in patients. [4, 5]. Additionally, the administration of topical glaucoma medications can lead to side effects, such as ocular irritation, decreasing the compliance of patients. These facts emphasize the importance of discovering new treatment strategies. The exact pathogenesis for glaucoma is still unknown, but several factors are considered to be involved (fig.1). The most prominent factor is an elevated IOP, which can cause blockade of axonal protein transport at the lamina cribrosa, causing an initial axonal damage and RGC death by trophic insufficiency. In addition, ischemic/hypoxic damage [6], astrocyte and glia cell alterations, and excessive stimulation of the glutamatergic system [7] are discussed as possible pathomechanisms. Moreover, an involvement of the immune system is considered [8-10]. In glaucoma patients, up- and downregulations in the systemic and ocular antibody profile were detected [11-13]. Also, antibody deposits were observed in glaucomatous retinae [14]. It is likely that a combination of several pathogenic factors/mechanisms increases the possibility of developing glaucoma. For the investigation of pathomechanisms and novel therapies, it is necessary to have suitable models that allow such screening. To investigate whether antibodies detected in glaucoma patients are part of glaucoma pathogenesis or a result of disease progression, the experimental autoimmune glaucoma (EAG) animal model was established. This animal model is based on the fact that autoantibodies against S100B, a small calcium binding protein expressed in glia cells, were found to be increased in glaucoma patients [15]. In this model, an immunization with S100B led to a significant loss of RGCs after 28 days and a fast degeneration of optic nerves [20]. The IOP in this model was not altered. Therefore, we were able to mimic the effects seen in NTG patients, an IOP-independent glaucomatous degeneration, by immunizing rats with S100B. S100B is a calcium-binding protein. In the central nervous system (CNS), it is mainly expressed by glial cells such as oligodendrocytes, Schwann cells, ependymal cells, retinal Müller cells and astrocytes [16]. S100B regulates and maintains the homeostasis of the important second messenger calcium and is therefore involved in many cell activities, such as signal transduction, cell differentiation, regulation of cell motility, transcription and cell cycle processes [17, 18]. Extracellularly, S100B can act as a signal molecule and bind receptors such as the receptor for advanced glycation end products (RAGE). In high concentrations, S100B can have negative effects and lead to cell death. For example, the binding of RAGE can induce the activation of microglia cells leading to a release of proinflammatory cytokines to an excessive extent [19]. Furthermore, there seems to be a link between S100B and neuronal diseases. In glaucoma patients, high antibody titers against S100B were found [20]. To investigate the effect of S100B in glaucoma more precisely, the immunization with S100B leads to a loss of retinal ganglion cells (RGCs) without an elevation of the IOP after 28 days in the EAG model [21, 22]. The goal of the present study was to detect the retinal biomarkers in EAG animals 7 and 14 days after immunization with S100B. To detect these new markers in disease development, a label free quantitative mass spectrometry-based approach will be used.

青光眼(Glaucoma)不仅是全球第二大致盲性眼病，且随着社会老龄化加剧，未来患者数量还将显著增长。青光眼是一种进行性视神经病变，特征为视神经乳头改变、视网膜神经节细胞(RGC)逐渐凋亡以及视野缺损[1]。

遗憾的是，青光眼在病程进展至较晚期前往往无明显症状，因此约10%~50%的患者并未察觉自己患病。高眼内压(IOP)是其主要危险因素，但正常眼压性青光眼(NTG)可发生于生理范围内眼内压的患者群体中[2]，该类型约占青光眼患者总数的30%[3]。此外，持续降低眼内压虽可延缓疾病进展，但无法完全阻止其发展[4,5]。同时，青光眼局部用药可能引发眼部刺激等不良反应，进而降低患者依从性。上述现状凸显了开发新型治疗策略的重要性。

青光眼的确切发病机制至今尚未明确，但已有多项因素被认为参与了疾病进程（fig.1）。其中最突出的致病因素为眼内压升高，其可导致筛板部位轴突蛋白运输受阻，引发初始轴突损伤，并通过营养不足途径导致视网膜神经节细胞凋亡。此外，缺血/缺氧损伤[6]、星形胶质细胞与胶质细胞异常、谷氨酸能系统过度激活[7]也被认为是潜在的发病机制。另有研究指出免疫系统亦参与其中[8-10]：在青光眼患者体内，已检测到全身及眼部抗体谱的上调与下调[11-13]，且在青光眼患者的视网膜中观察到抗体沉积现象[14]。多种致病因素/机制的协同作用，大概率增加了青光眼的发病风险。

为研究青光眼的发病机制并探索新型治疗方案，需建立适用于相关筛选的合适模型。为验证青光眼患者体内检测到的抗体是疾病发病机制的一部分，还是疾病进展的结果，研究者构建了实验性自身免疫性青光眼(EAG)动物模型。该模型基于“青光眼患者体内针对S100B的自身抗体水平升高”这一发现——S100B是一种在胶质细胞中表达的小型钙结合蛋白[15]。在此模型中，用S100B进行免疫接种可在28天后引发显著的视网膜神经节细胞丢失，并加速视神经退行性变[20]，且该模型中的眼内压并未发生改变。因此，通过向大鼠接种S100B，我们成功模拟了正常眼压性青光眼患者所表现出的、非眼内压依赖型的青光眼性退行性变。

S100B是一种钙结合蛋白。在中枢神经系统(CNS)中，它主要由少突胶质细胞、施万细胞、室管膜细胞、视网膜米勒细胞及星形胶质细胞等胶质细胞表达[16]。S100B可调节并维持重要第二信使钙离子的稳态，因此参与诸多细胞活动，包括信号转导、细胞分化、细胞运动调控、转录及细胞周期进程[17,18]。在细胞外环境中，S100B可作为信号分子结合晚期糖基化终末产物受体(RAGE)等受体。当浓度过高时，S100B可产生负面效应并引发细胞死亡：例如，其与RAGE结合可诱导小胶质细胞活化，进而过度释放促炎细胞因子[19]。此外，S100B似乎与神经系统疾病存在关联：在青光眼患者体内，已检测到抗S100B抗体滴度升高[20]。

为更精准地探究S100B在青光眼发病中的作用，已有研究证实，在EAG模型中，用S100B免疫接种可在28天后引发视网膜神经节细胞丢失，且不伴随眼内压升高[21,22]。本研究的目标为：在使用S100B免疫接种后的第7天和第14天，检测EAG动物体内的视网膜生物标志物。为捕捉疾病发展过程中的新型标志物，本研究将采用无标记定量质谱技术进行分析。